There are a multitude of techniques employed for the quantitative determination of an unknown quantity of a sample derived from a biological fluid (e.g., serum or urine). Techniques based upon radioactive labelled substance include indirect (competitive binding, saturation or displacement) and direct (sandwich and immunoradiometric) radioimmunassays. Other known techniques are based on fluorochrome and enzyme labels. After completion of the above reactions, the labelled substances which have been bound must be separated from the unreacted labelled substance which includes free and non-specifically bound substance. This separation step in liquid form can be inefficient, unreliable and inconvenient.
A number of techniques have been proposed to solve this problem by use of diagnostic reagents on solid surfaces which combine with the labelled substance. In each of these systems, the solid substrate containing the bound labelled substance is separated from the liquid phase containing the free labelled substance and is thereafter washed to remove the residual free and the non-specifically bound labelled substance.
In one technique for the detection of antigen, reagents are coated upon plastic test tubes or inserts by the physical adsorption of antibodies specific to the sample substances to be tested. Such techniques are disclosed in U.S. Pat. Nos. 3,646,346 and 3,826,619. Such physical adsorption is difficult to control because of the non-uniformity of plastic surfaces as well as imprecision in the coating techniues. See, e.g., the work entitled "Radioimmunoassay Methods", European Workshop, Sept. 15-17, 1970, Edinburgh (Editors Kirkham et al). At page 441, the authors state that the amount of binding is between 20% and 70% using various coated polystyrene tubes in a particular system, which led to the abandonment of this technique. Furthermore, in subsequent immunochemical reactions, a significant amount of "non-specific" binding of the labelled substance to the solid surface occurs. This binding, of a hydrophobic or ionic nature, is weaker than the immunochemical bond. Hence, vigorous and repeated washings required to effectively remove solution residual and non-specifically bound labelled substance frequently disrupts the weaker physical coated bond resulting in the loss of physically bound diagnostic reagent. The results of assays employing such coated surfaces are thus relatively imprecise and non-reproducible because of these kinds of misclassification errors. (Envall et al., Biochim. Biophys. Acta, 251 (1971) 427-434, at p. 430). Another disadvantage of physically coating as with antibodies is that the antibodies in that form are relatively unreactive with large antigens. It is believed that this is due to steric hindrance effects.
In the technique of Bennich et al U.S. Pat. No. 3,720,760, immunological substances are more firmly secured by attachment to porous microreticular swellable polymer of the type sold under the trademark "Sephadex". Such material is indicated as being preferably in particulate form. Such microreticular materials have the inherent disadvantage of being difficult to wash their interstitial void spaces free of non-specifically bound and entrained free labelled substance. This results in undesirably high background readings. Also, it is difficult to rinse the interparticulate void spaces sufficiently free of residual free labelled substances, generally requiring several displacement washes. Each washing step requires a centrifugal and careful decantation separation to avoid misclassification to minimize inadvertent spillage of particles during the washing procedure.
This technique is unsuitable in fluorometric and colorimetric surface systems because the particles cannot be accurately viewed. This is because loose microreticular beads used in radioactive assays produce an ill-defined and difficult to control surface for uniform illumination and viewing.
Another technique which has been proposed is described in an article entitled "Solid Phase Radioimmunoassay of Human Growth Hormone" by Catt et al, J. Lab. & Clin. Med., (1966) 100, 31c. Small discs of substituted graft copolymer sold under the trademark "Protapol DI/1", are coated with antibody. As set forth in the above Kirkham et al work, at pages 294 and 295, this technique yields poor replication, i.e., there is a very wide scatter between the replicates at any given point, and sometimes the differences are very large. The discs require repeated handling and each time they are touched by a solid object the chance for loss of bound labelled substance or contamination by free labelled substance or other background contributing contaminants is considerable. Like the above particle technique, the disc would not be accurately viewed in fluorometric or colorimetric systems and would be difficult to manipulate into proper viewing relationship with an optical instrument.
In view of the foregoing, there is an apparent need for a solid phase analytical technique in which the surface is easy to wash, separations are easy to make, which is efficient and reproducible, and which can be presented easily for accurate viewing.